Team:Imperial College London/Wetlab/Protocols

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| Colanic Acid  
| Colanic Acid  
| Colanic acid biosynthesis is both time consuming and metabolically expensive. If the E.ncapsulator is to be used in an industrial setting, then colanic acid mediated protection must be highly efficient. Protective efficiency can be defined as the percentage increase in fluorescence per µl of colanic acid produced (when compared to control cells). The following assay can be used to elucidate this parameter.
| Colanic acid biosynthesis is both time consuming and metabolically expensive. If the E.ncapsulator is to be used in an industrial setting, then colanic acid mediated protection must be highly efficient. Protective efficiency can be defined as the percentage increase in fluorescence per µl of colanic acid produced (when compared to control cells). The following assay can be used to elucidate this parameter.
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| 3.Genome Deletion
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| [[https://2009.igem.org/Team:Imperial_College_London/Wetlab/Protocols/Restriction |In Vitro Restriction]]
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| * By running restriction digests on the genome of E.coli strains, we can investigate the efficiency of our restriction enzyme, taqI and dpnII, on genome deletion.
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* Run parallel digests with our chassis of choice (TOP10) and a Dam negative control (of which the DNA should not be methylated) to investigate the effects of methylation on cleavage.
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|- style="color:#333; background-color:#CCCCFF;" cellpadding="6" cellspacing="0" border="1"

Revision as of 11:40, 18 September 2009



Contents

Protocols

  1. Calibrations
    • Top 10 Growth Calibration
  2. Promoter Characterisation
    • Lac Promoter
  3. Auto Induction
  4. Protein Production
  5. Colanic Acid Encapsulation
  6. Trehalose Production
  7. Thermoinduction
  8. Genome Restriction


Assay

  • The cells will be grown until OD= 0.7.
  • The RFP value will be monitered, although it is the GFP values that are more critical in this assay.
  • OD and fluorescence data for GFP which can be converted in [http://partsregistry.org/cgi/measurement/new_batch.cgi Specific Promoter Units (SPUs)].
  • The secondary carbon source will be taken from the previous experiment (see Secondary carbon source selection for CRP promoter)
  • The IPTG concentrations will be taken from the previous experiment (see Determining concentration of IPTG)

Team:Imperial_College_London/Wetlab/See Glucose time delay for details



Section Assay Overview and Aims
1.Calibration TOP10 Growth * To produce a calibration curve to aid in the normalising of absorbance values. The relation of absorbance reading to number of cells varies with different cell strains. We are therefore doing one for Top-10.


1.Promoter Characterisation Blah To find
2.Auto-Induction Secondary Carbon Source Diauxie Growth
  • To generate the diauxie growth curves, as well as normal growth curves for Top-10 for modelling
  • To determine the best secondary carbon source for optimal growth
2.Auto-Induction Glucose Time Delay * Characterise the tunable time duration it takes before GFP expression (M2 activation)
3.Protein Production IPTG Toxicity To investigate the effect of our IPTG inducer on growth of our cultures.
  • Measure growth rates at 28 degrees Celsius on minimal growth media so that subsequent testing timings etc. can be streamlined
  • Determine the effect of IPTG toxicity on growth w/o any protein production complications
3.Protein Production Cellulase Aims
3.Protein Production PAH Aims
4.Colanic Acid Encapsulation Colanic Acid Colanic acid biosynthesis is both time consuming and metabolically expensive. If the E.ncapsulator is to be used in an industrial setting, then colanic acid mediated protection must be highly efficient. Protective efficiency can be defined as the percentage increase in fluorescence per µl of colanic acid produced (when compared to control cells). The following assay can be used to elucidate this parameter. 3.Genome Deletion [|In Vitro Restriction] * By running restriction digests on the genome of E.coli strains, we can investigate the efficiency of our restriction enzyme, taqI and dpnII, on genome deletion.
  • Run parallel digests with our chassis of choice (TOP10) and a Dam negative control (of which the DNA should not be methylated) to investigate the effects of methylation on cleavage.

Promoter characterisation assays

Absorbance calibration

Aim

  • To produce a calibration curve to aid in the normalising of absorbance values. The relation of absorbance reading to number of cells varies with different cell strains. We are therefore doing one for Top-10.

Assay

Cultures of the E.coli with the relevant vector are grown to various cell densities. A sample of these cultures are taken and a dilution plate is carried out to work out approximate colony forming units per ml of culture. This data set is then combined with absorbance readings to create a graph relating the number of colony forming cells per ml to their absorbance measurements.

This curve then allows us to convert absorbance of a known volume of culture to colony forming units within the culture sample.

See the protocol for more details

Autoinduction assays

IPTG effect on growth

Aims

  • Measure growth rates at 28 degrees Celsius on minimal growth media so that subsequent testing timings etc. can be streamlined
  • Determine the effect of IPTG toxicity on growth w/o any protein production complications

Assay

Normal cells (without any constructs) will be grown on M9 media until OD=0.7.
IPTG of various concentrations will then be added, and the OD of the cells will be followed over time


See the protocol for more details

Lac characterisation and IPTG effect on protein production

Aims

  • Characterise Lac promoter by varying amounts of IPTG
  • Determine the IPTG concentration that allows for maximal protein production while still being non-toxic to the cell

Assay

The cells will be grown until OD=0.7. Now, IPTG of various concentrations will be added, and the RFP output will be measured.
The experiment will generate OD and fluoresence data for RFP
The secondary carbon source will be taken from the previous experiment (see Secondary carbon source selection)
The glucose concentration will be taken from commercial autoinduction media (0.05%) – takes about 7 hours to exhaust

See the protocol for more details

Mr. Gene   Geneart   Clontech   Giant Microbes

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